Figure 4.

Omission of the RNaseR based circRNA enrichment steps decreases the bias against longer circRNAs. (A) Plot showing the ratios of the circRNA read counts of the circ-enriched IMCR-seq protocol and the total RNA IMCR-seq protocol of different circRNA sizes. Error bars represent the standard error of the mean. (B) Plot showing the mean amounts of a linear RNA, a medium-size (789 nt) circRNA, and a large-size (1,742 nt) circRNA detected with Agilent TapeStation after in vitro RNaseR digestion at 30 and 60 minutes. For normalization, corresponding RNAs that were incubated at 37°C for 60 minutes in 1× reaction conditions without RNaseR were used (n = 3, *P= 0.023 calculated via one-sample, two-tailed t-test; ** P= 0.001 calculated via paired, two-tailed t-test). (C) Schematic showing experimental confirmation of long circRNAs detected with total IMCR-seq with divergent primers spanning the BSJ (a 7,166 nt-long CHIC1 circRNA isoform is illustrated here as an example, a similar divergent primer PCR strategy was followed for the other long circRNAs in Supplementary Figure S8). (D) Sanger sequencing of 7,166 nt CHIC1 circRNA isoform BSJ region.